Interleukin-12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor or natural killer cell stimulatory factor, is a 75-KDa heterodimeric cytokine composed of disulfide-bonded 40-KDa (p40) and -KDa (p35) subunits that has pleiotropic activities including stimulation of the proliferation of activated T and NK cells (Gately, M. K., et al., 1991, J. Immunol., 147:874) (Kobayashi, M., et al., 1989, J. Exp. Med., 170:827), enhancement of the lytic activity of NK/LAK cells (Kobayashi, M., et al., supra) (Stern, A. S., et al., 1990, Proc. Natl. Acad. Sci. USA, 87:6808), enhancement of cytolytic T-cell responses (M. Gately et al., 1992, Cell. Immunology, 143:127), induction of interferon gamma by resting and activated T- and NK-cells (M. Kobayashi et al., supra; S. H. Chan et al., 1991, J. Exp. Med., 173:869), and promotion of T.sub.h 1-type helper cell responses (R. Manetti et al., 1993, J. Exp. Med., 177:1199; C. S. Hsieh et al., 1993, Science 260:547).
The biological activity of IL-12 is mediated by the binding of the IL-12 molecules to cell surface, or plasma membrane, receptors on activated T- and NK cells; however, the contributions of the individual subunits, p35 and p40, to receptor binding and signal transduction remain unknown. Studies with labeled IL-12 have shown that this binding occurs in a specific and saturable manner. IL-12 delivers a signal to target cells through a receptor that was initially characterized on PHA-activated CD4+ and CD8+ T-cells and on IL-2 activated CD56+ NK-cells (R. Chizzonite et al., 1992, J. Immunol., 148:3117; B. Desai, et al., 1992, J. Immunol., 148:3125). A survey of over 20 human cell lines belonging to the T-, B-, NK- and myelomonocytic lineages only identified a single CD4+, IL-2 dependent human T-cell line (Kit 225) that constitutively expresses the IL-12 receptor and responds to IL-12 (B. Desai, et al., 1992, J. Immunol., 148:3125; B. Desai, et al., 1993, J. Immunol. 150:207A). Freshly prepared PHA-activated PBMC and the Kit 225 cell line thus represent two convenient cell sources to study the biochemistry of the functional IL-12 receptor; there may be others. Equilibrium binding experiments with .sup.125 I-labeled IL-12 showed that i) PHA-activated PBMC express several thousand IL-12 receptors which show 3 classes of affinities: high =5-20 pM, intermediate =50-200 pM and low =2-6 nM; ii) IL-12 receptor expression on PBMC is upregulated by mitogen or IL-2 stimulation; and iii) the IL-12 receptor upregulation correlates with the ability of the cells to proliferate in response to IL-12 (R. Chizzonite, et al., 1992, J. Immunol., 148:3117; B. Desai, et al., 1992, J. lmmunol., 148:3125). It was not clear at this point whether the biologically functional IL-12 receptor consists of one or more subunits. Affinity crosslinking of labeled IL-12 to activated PBMC demonstrated I 0 the size of the cell surface IL-12 binding protein(s) under nonreducing conditions to be in the range of about 150 KDa to about 200 KDa. Additional affinity crosslinking and immunoprecipitation experiments with unlabeled IL-12 bound to .sup.125 I-surface labeled activated PBMC identified an IL-12 binding protein that under reducing conditions had a size of about 110 KDa (R. Chizzonite, et al., 1992, J. Immunol., 148:3117).
Using a non-neutralizing monoclonal antibody to the IL-12 receptor, we have now succeeded in isolating a human eDNA that encodes a low affinity (5-10 nM) IL-12 receptor. This protein belongs to the cytokine receptor superfamily and within that family shows strongest homology to gp130.
In order for a molecule such as IL-12 to exert its effect on cells, it is now accepted by those skilled in the art that the molecule must interact with molecules, located on cell membranes, referred to as receptors. Patents which exemplify disclosures of interleukin receptors include Honjo et al., U.S. Pat. No. 4,816,565; Urdal et al., U.S. Pat. No. 4,578,335; Dower et al., U.S. Pat. No. 5,180,812; and Taniguchi et al., U.S. Pat. No. 5,198,359, the disclosures of which are incorporated by reference.
Fanslow, W. C. et al., Science 248:739-41 (May 11, 1990) presented data showing that the effect of IL-1 in vivo could be regulated via the administration of a soluble form of its receptor. The results that Fanslow report demonstrate the ability of a soluble cytokine receptor (soluble IL-1R) to modulate biological activity upon exogeneous administration in vivo, presumably by acting as a neutralizing agent for the endogeneously produced, corresponding ligand (IL-1), and provides evidence of the therapeutic potential of soluble cytokine receptors in a variety of clinical disorders. Systemic administration of a soluble, extracellular portion of the receptor for IL-1 (soluble IL-1R) had profound inhibitory effects on the development of in vivo alloreactivity. Survival of heterotopic heart allografts was prolonged from 12 days in controls to 17 days in mice treated with soluble IL-1R. Lymph node hyperplasia in response to localized injection of allogeneic cells was completely blocked by soluble IL- 1R treatment. What types of therapeutic efficacy that administration of soluble IL-12 receptor is expected to have can also be contemplated therefor by those skilled in the art.
The availability of the purified receptor, in soluble form, presents therapeutic possibilities as well, as shown by Fanslow above. Addition of soluble IL-12 receptor interferes with the effect of the interleukin on the cells, since the molecule cannot bind to the cell membrane as freely. Hence, an aspect of the invention is the treatment of pathological conditions caused by excess activity of cells possessing IL-12 receptors by adding an amount of soluble IL-12 receptors sufficient to inhibit binding of IL-12 to the aforementioned cells. This methodology can also be modified, and the soluble receptor can also be used as a screening agent for pharmaceuticals. Briefly, a pharmaceutical which works as an IL-12 antagonist can do so by blocking the binding of IL-12 to the IL-12 receptor. Prior to determining whether a material would be effective in vivo, one may use the purified IL-12 receptor in connection with a potential pharmaceutical to determine if there is binding. If not, then the pharmaceutical may no longer be a desirable candidate. If there is in fact binding, further testing may be indicated.